MoCDC14 is important for septation during conidiation and appressorium formation in Magnaporthe oryzae

As a typical foliar pathogen, appressorium formation and penetration are critical steps in the infection cycle of Magnaporthe oryzae. Because appressorium formation and penetration are closely co‐regulated with the cell cycle, and Cdc14 phosphatases have an antagonistic relationship with cyclin‐dependent kinases (CDKs) on proteins related to mitotic exit and cytokinesis, in this study, we functionally characterized the MoCDC14 gene in M. oryzae. The Mocdc14 deletion mutant showed significantly reduced growth rate and conidiation. It was also defective in septum formation and nuclear distribution. Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi‐nucleate. Mutant conidia often showed incomplete septa or lacked any septum. During appressorium formation, the septum delimiting appressoria from the rest of the germ tubes was often formed far away from the neck of the appressoria or not formed at all. Unlike the wild‐type, some mutant appressoria had more than one nucleus at 24 h. In addition to appressoria, melanization occurred on parts of the germ tubes and conidia, depending on the irregular position of the appressorium‐delimiting septum. The Mocdc14 mutant was also defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells. Similar defects in septum formation, melanization and penetration were observed with appressorium‐like structures formed at hyphal tips in the Mocdc14 mutant. Often a long fragment of mutant hyphae was melanized, together with the apical appressorium‐like structures. These results indicate that MoCDC14 plays a critical role in septation, nuclear distribution and pathogenesis in M. oryzae, and correct septum formation during conidiogenesis and appressorium formation requires the MoCdc14 phosphatase.     

The RasGEF FgCdc25 regulates fungal development and virulence in Fusarium graminearum via cAMP and MAPK signalling pathways

Ras GTPases act as molecular switches to control various cellular processes by coupling integrated signals in eukaryotes. Activities of Ras GTPases are triggered by Ras GTPase guanine nucleotide exchange factors (RasGEFs) in general, whereas the role of RasGEF in plant pathogenic fungi is largely unknown. In this study, we characterized the only RasGEF protein in Fusarium graminearum, FgCdc25, by combining genetic, cytological and phenotypic strategies. FgCdc25 directly interacted with RasGTPase FgRas2, but not FgRas1, to regulate growth and sexual reproduction. Mutation of the FgCDC25 gene resulted in decreased toxisome formation and deoxynivalenol (DON) production, which was largely depended on cAMP signalling. In addition, FgCdc25 indirectly interacted with FgSte11 in FgSte11-Ste7-Gpmk1 cascade, and the ΔFgcdc25 strain totally abolished the formation of infection structures and was nonpathogenic in planta, which was partially recovered by addition of exogenous cAMP. In contrast, FgCdc25 directly interplayed with FgBck1 in FgBck1-MKK1-Mgv1 cascade to negatively control cell wall integrity. Collectively, these results suggest that FgCdc25 modulates cAMP and MAPK signalling pathways and further regulates fungal development, DON production and plant infection in F. graminearum.     

Dephosphorylation of Iqg1 by Cdc14 regulates cytokinesis in budding yeast

Cytokinesis separates cells by contraction of a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential protein in Saccharomyces cerevisiae required for assembly and contraction of the actomyosin ring. Localization of F-actin to the ring occurs only after anaphase and is mediated by the calponin homology domain (CHD) of Iqg1, but the regulatory mechanisms that temporally restrict actin ring assembly are not well defined. We tested the hypothesis that dephosphorylation of four perfect cyclin-dependent kinase (Cdk) sites flanking the CHD promotes actin ring formation, using site-specific alanine mutants. Cells expressing the nonphosphorylatable iqg1-4A allele formed actin rings before anaphase and exhibited defects in myosin contraction and cytokinesis. The Cdc14 phosphatase is required for normal cytokinesis and acts on specific Cdk phosphorylation sites. Overexpression of Cdc14 resulted in premature actin ring assembly, whereas inhibition of Cdc14 function prevented actin ring formation. Cdc14 associated with Iqg1, dependent on several CHD-flanking Cdk sites, and efficiently dephosphorylated these sites in vitro. Of importance, the iqg1-4A mutant rescued the inability of cdc14-1 cells to form actin rings. Our data support a model in which dephosphorylation of Cdk sites around the Iqg1 CHD by Cdc14 is both necessary and sufficient to promote actin ring formation. Temporal control of actin ring assembly by Cdk and Cdc14 may help to ensure that cytokinesis onset occurs after nuclear division is complete.     

Dependence of Chs2 ER export on dephosphorylation by cytoplasmic Cdc14 ensures that septum formation follows mitosis

Cytokinesis, which leads to the physical separation of two dividing cells, is normally restrained until after nuclear division. In Saccharomyces cerevisiae, chitin synthase 2 (Chs2), which lays down the primary septum at the mother-daughter neck, also ensures proper actomyosin ring constriction during cytokinesis. During the metaphase-to-anaphase transition, phosphorylation of Chs2 by the mitotic cyclin-dependent kinase (Cdk1) retains Chs2 at the endoplasmic reticulum (ER), thereby preventing its translocation to the neck. Upon Cdk1 inactivation at the end of mitosis, Chs2 is exported from the ER and targeted to the neck. The mechanism for triggering Chs2 ER export thus far is unknown. We show here that Chs2 ER export requires the direct reversal of the inhibitory Cdk1 phosphorylation sites by Cdc14 phosphatase, the ultimate effector of the mitotic exit network (MEN). We further show that only Cdc14 liberated by the MEN after completion of chromosome segregation, and not Cdc14 released in early anaphase by the Cdc fourteen early anaphase release pathway, triggers Chs2 ER exit. Presumably, the reduced Cdk1 activity in late mitosis further favors dephosphorylation of Chs2 by Cdc14. Thus, by requiring declining Cdk1 activity and Cdc14 nuclear release for Chs2 ER export, cells ensure that septum formation is contingent upon chromosome separation and exit from mitosis.     

Cdk1 phosphorylation of fission yeast paxillin inhibits its cytokinetic ring localization

Divisions of the genetic material and cytoplasm are coordinated spatially and temporally to ensure genome integrity. This coordination is mediated in part by the major cell cycle regulator cyclin-dependent kinase (Cdk1). Cdk1 activity peaks during mitosis, but during mitotic exit/cytokinesis Cdk1 activity is reduced, and phosphorylation of its substrates is reversed by various phosphatases including Cdc14, PP1, PP2A, and PP2B. Cdk1 is known to phosphorylate several components of the actin- and myosin-based cytokinetic ring (CR) that mediates division of yeast and animal cells. Here we show that Cdk1 also phosphorylates the Schizosaccharomyces pombe CR component paxillin Pxl1. We determined that both the Cdc14 phosphatase Clp1 and the PP1 phosphatase Dis2 contribute to Pxl1 dephosphorylation at mitotic exit, but PP2B/calcineurin does not. Preventing Pxl1 phosphorylation by Cdk1 results in increased Pxl1 levels, precocious Pxl1 recruitment to the division site, and increased duration of CR constriction. In vitro Cdk1-mediated phosphorylation of Pxl1 inhibits its interaction with the F-BAR domain of the cytokinetic scaffold Cdc15, thereby disrupting a major mechanism of Pxl1 recruitment. Thus, Pxl1 is a novel substrate through which S. pombe Cdk1 and opposing phosphatases coordinate mitosis and cytokinesis.     

Dual Regulation of the Mitotic Exit Network (MEN) by PP2A-Cdc55 Phosphatase

Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2A^Cdc55 phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2A^Cdc55 phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2A^Cdc55 regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2A^Cdc55 initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2A^Cdc55 functions affect the regulation of various MEN components, contributing to mitotic exit.     

Two Cdc2 Kinase Genes with Distinct Functions in Vegetative and Infectious Hyphae in Fusarium graminearum

Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.     

FgTep1p is linked to the phosphatidylinositol‐3 kinase signalling pathway and plays a role in the virulence of Fusarium graminearum on wheat

Both mammalian tensin‐like phosphatase 1 [TEP1; also known as phosphatase deleted on chromosome 10 (PTEN) or mutated in multiple advanced cancer 1 (MMAC1)] and Saccharomyces cerevisiae ScTep1p are involved in the phosphatidylinositol pathway. In this study, we identified the Fusarium graminearum locus FGSG_04982.3 (named FgTEP1) as the functional homologue of ScTEP1 in the sensitivity of S. cerevisiae cells to wortmannin, the phosphatidylinositol‐3 kinase inhibitor. Deletion of FgTEP1 causes F. graminearum mycelial growth to become sensitive to lithium and reduces the production of conidia. Although conidia lacking FgTEP1 germinate normally, they show reduced germination efficiency in the presence of wortmannin. In addition, we showed that deletion of FgTEP1 reduces the virulence of F. graminearum on wheat. These results indicate that FgTep1p is linked to the phosphatidylinositol‐3 kinase signalling pathway in this plant fungal pathogen.     

Meiotic nuclear divisions in budding yeast require PP2A(Cdc55)-mediated antagonism of Net1 phosphorylation by Cdk

During meiosis, one round of deoxyribonucleic acid replication is followed by two rounds of nuclear division. In Saccharomyces cerevisiae, activation of the Cdc14 early anaphase release (FEAR) network is required for exit from meiosis I but does not lead to the activation of origins of replication. The precise mechanism of how FEAR regulates meiosis is not understood. In this paper, we report that premature activation of FEAR during meiosis caused by loss of protein phosphatase PP2A(Cdc55) activity blocks bipolar spindle assembly and nuclear divisions. In cdc55 meiotic null (cdc55-mn) cells, the cyclin-dependent kinase (Cdk)-counteracting phosphatase Cdc14 was released prematurely from the nucleolus concomitant with hyperphosphorylation of its nucleolar anchor protein Net1. Crucially, a mutant form of Net1 that lacks six Cdk phosphorylation sites rescued the meiotic defect of cdc55-mn cells. Expression of a dominant mutant allele of CDC14 mimicked the cdc55-mn phenotype. We propose that phosphoregulation of Net1 by PP2A(Cdc55) is essential for preventing precocious exit from meiosis I.     

Separase cooperates with Zds1 and Zds2 to activate Cdc14 phosphatase in early anaphase

Completion of mitotic exit and cytokinesis requires the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. A key enzyme that counteracts Cdk during budding yeast mitotic exit is the Cdc14 phosphatase. Cdc14 is inactive for much of the cell cycle, sequestered by its inhibitor Net1 in the nucleolus. At anaphase onset, separase-dependent down-regulation of PP2A^Cdc55 allows phosphorylation of Net1 and consequent Cdc14 release. How separase causes PP2A^Cdc55 down-regulation is not known. Here, we show that two Cdc55-interacting proteins, Zds1 and Zds2, contribute to timely Cdc14 activation during mitotic exit. Zds1 and Zds2 are required downstream of separase to facilitate nucleolar Cdc14 release. Ectopic Zds1 expression in turn is sufficient to down-regulate PP2A^Cdc55 and promote Net1 phosphorylation. These findings identify Zds1 and Zds2 as new components of the mitotic exit machinery, involved in activation of the Cdc14 phosphatase at anaphase onset. Our results suggest that these proteins may act as separase-regulated PP2A^Cdc55 inhibitors.     

The Sch9 Kinase Regulates Conidium Size,Stress Responses,and Pathogenesis in Fusarium graminearum

Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley worldwide. In a previous study on functional characterization of the F. graminearum kinome, one protein kinase gene important for virulence is orthologous to SCH9 that is functionally related to the cAMP-PKA and TOR pathways in the budding yeast. In this study, we further characterized the functions of FgSCH9 in F. graminearum and its ortholog in Magnaporthe oryzae. The ΔFgsch9 mutant was slightly reduced in growth rate but significantly reduced in conidiation, DON production, and virulence on wheat heads and corn silks. It had increased tolerance to elevated temperatures but became hypersensitive to oxidative, hyperosmotic, cell wall, and membrane stresses. The ΔFgsch9 deletion also had conidium morphology defects and produced smaller conidia. These results suggest that FgSCH9 is important for stress responses, DON production, conidiogenesis, and pathogenesis in F. graminearum. In the rice blast fungus Magnaporthe oryzae, the ΔMosch9 mutant also was defective in conidiogenesis and pathogenesis. Interestingly, it also produced smaller conidia and appressoria. Taken together, our data indicate that the SCH9 kinase gene may have a conserved role in regulating conidium size and plant infection in phytopathogenic ascomycetes.     

The nucleolar phosphatase Cdc14B is dispensable for chromosome segregation and mitotic exit in human cells

In yeast, the protein phosphatase Cdc14 promotes chromosome segregation, mitotic exit, and cytokinesis by reversing M-phase phosphorylations catalyzed by Cdk1. A key feature of Cdc14 regulation is its sequestration within the nucleolus, which restricts its access to potential substrates for much of the cell cycle. Mammals also possess a nucleolar Cdc14 homolog, termed Cdc14B, but its roles during mitosis and cell division remain speculative. Here we analyze Cdc14B’s subcellular dynamics during mitosis and rigorously test its functional contributions to cell division through homozygous disruption of the Cdc14B locus in human somatic cells. While Cdc14B is initially released from nucleoli at the start of mitosis, the phosphatase quickly redistributes onto segregating sister chromatids during anaphase. This relocalization is mainly driven by Cdk1 inactivation, as pharmacologic inhibition of Cdk1 in prometaphase cells redirects Cdc14B onto chromosomes. However, in sharp contrast to yeast cdc14 mutants, human Cdc14BΔ/Δ cells were viable and lacked defects in spindle assembly, anaphase progression, mitotic exit, and cytokinesis, and continued to segregate ribosomal DNA repeats with near-normal proficiency. Our findings reveal substantial divergence in mitotic regulation between yeast and mammalian cells, as the latter possess efficient mechanisms for completing late M-phase events in the absence of a nucleolar Cdc14-related phosphatase.     

Protein kinase FgSch9 serves as a mediator of the target of rapamycin and high osmolarity glycerol pathways and regulates multiple stress responses and secondary metabolism in Fusarium graminearum

Saccharomyces cerevisiae protein kinase Sch9 is one of the downstream effectors of the target of rapamycin (TOR) complex 1 and plays multiple roles in stress resistance, longevity and nutrient sensing. However, the functions of Sch9 orthologs in filamentous fungi, particularly in pathogenic species, have not been characterized to date. Here, we investigated biological and genetic functions of FgSch9 in Fusarium graminearum. The FgSCH9 deletion mutant (ΔFgSch9) was defective in aerial hyphal growth, hyphal branching and conidial germination. The mutant exhibited increased sensitivity to osmotic and oxidative stresses, cell wall‐damaging agents, and to rapamycin, while showing increased thermal tolerance. We identified FgMaf1 as one of the FgSch9‐interacting proteins that plays an important role in regulating mycotoxin biosynthesis and virulence of F. graminearum. Co‐immunoprecipitation and affinity capture‐mass spectrometry assays showed that FgSch9 also interacts with FgTor and FgHog1. More importantly, both ΔFgSch9 and FgHog1 null mutant (ΔFgHog1) exhibited increased sensitivity to osmotic and oxidative stresses. This defect was more severe in the FgSch9/FgHog1 double mutant. Taken together, we propose that FgSch9 serves as a mediator of the TOR and high osmolarity glycerol pathways, and regulates vegetative differentiation, multiple stress responses and secondary metabolism in F. graminearum.     

The FRP1 F-box gene has different functions in sexuality, pathogenicity and metabolism in three fungal pathogens

Plant‐pathogenic fungi employ a variety of infection strategies; as a result, fungi probably rely on different sets of proteins for successful infection. The F‐box protein Frp1, only present in filamentous fungi belonging to the Sordariomycetes, Leotiomycetes and Dothideomycetes, is required for nonsugar carbon catabolism and pathogenicity in the root‐infecting fungus Fusarium oxysporum. To assess the role of Frp1 in other plant‐pathogenic fungi, FRP1 deletion mutants were generated in Fusarium graminearum and Botrytis cinerea, and their phenotypes were analysed. Deletion of FgFRP1 in F. graminearum led to impaired infection of barley roots, but not of aerial plant parts. Deletion of BcFRP1 in B. cinerea did not show any effect on pathogenicity. Sexual reproduction, however, was impaired in both F. graminearum and B. cinerea FRP1 deletion mutants. The mutants of all three fungi displayed different phenotypes when grown on an array of carbon sources. The F. oxysporum and B. cinerea deletion mutants showed opposite growth phenotypes on sugar and nonsugar carbon sources. Replacement of FoFRP1 in F. oxysporum with the B. cinerea BcFRP1 resulted in the restoration of pathogenicity, but also in a switch from impaired growth on nonsugar carbon sources to impaired growth on sugar carbon sources. This effect could be ascribed in part to the B. cinerea BcFRP1 promoter sequence. In conclusion, the function of the F‐box protein Frp1, despite its high sequence conservation, is not conserved between different fungi, leading to differential requirements for pathogenicity and carbon source utilization.     

Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.